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Abstract

Inositol triphosphate (IP3) is an important second messenger that participates in signal transduction pathways in diverse cell types including hippocampal neurons. Stimulation of phospholipase C in response to various stimuli (hormones, growth factors, neurotransmitters, neurotrophins, neuromodulators, odorants, light, etc.) results in hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, and leads to the production of IP3 and diacylglycerol. Binding of IP3 to the IP3 receptor (IP3R) induces Ca2+ release from intracellular stores and enables the initiation of intracellular Ca2+-dependent signaling. Here we describe a procedure for the measurement of cellular IP3 levels in tissue homogenates prepared from rat hippocampal slices.

Place ~ 200 ml s-aCSF per rat at
-80 °C for approximately 40 min to form a slush (if s-aCSF is
pre-chilled at 4 °C slush will be formed more quickly, in 15-20 min).

Place Netwell insert into a glass staining dish with 350 ml regular
aCSF (constantly oxygenating with 95% O2/5% CO2 mixture using a coarse
glass bubbler) and put the dish into a water bath at 34-35 °C 20 min
prior to slice preparation.

Pour 20-30 ml s-aCSF slush in a
small glass beaker, place the beaker on ice and oxygenate with 95% O2/5%
CO2 mixture using a coarse glass bubbler for 10-15 min.

Pre-chill the microtome stage by placing ice in the ice chamber and
attach the double edge razor blade to the blade holder (Figure 2A).

Bend the tip of a 27 gauge needle (~45 degrees) using forceps and place it on a 1 ml syringe.

Deeply anesthetize rats with sodium pentobarbital (125 mg/kg, i.p.)
and perform laparotomy using surgical scissors and forceps until the
heart is completely exposed. Clamp the tip of the sternum with a
hemostat and place the hemostat over the head.

Remove the head immediately, quickly make an incision in the middle
of the scalp, pull aside the skin and cut through the skull along the
midline suture of the parietal plates. Remove the skull plates,
carefully extract the brain using a spatula and transfer it into
oxygenated and pre-chilled s-aCSF prepared in the step A1c for about one
minute. Note: Estimated time to carry out step A4 is less than a
minute.

Place the brain on a glass plate on ice (Figure 1A).
Hemisect the brain along the longitudinal fissure (Figure 1A, dashed
line 1) and make two 45 degree angle cuts relative to the coronal plane
(Figure 1A, dashed lines 2 and 3) for each hemisphere, producing tissue
blocks containing the hippocampus (Figure 1B).

Figure 1. Preparation of the brain for slicing. A.
Perfused rat brain on a glass plate on ice. The dashed lines indicate
where cuts are made and numbers indicate the order of the cuts. B.
Blocked brain with the two hemispheres separated. C. One hemisphere of
the brain mounted on the mounting disk of a Leica VT1200S vibrating
microtome.

Place a small drop of super glue in the center of
the mounting disk, carefully place one hemisphere on the drop of glue
with rostral side up (Figure 1C) and transfer to the sectioning stage of
the vibrating microtome (Figure 2A).

Transfer the sections into separate wells of the Netwell insert
(Figure 3A) using a disposable transfer pipette and let the sections
recover at 34-35 °C in oxygenated regular aCSF for 30-60 min. Make sure
the sections are resting flat on the mesh of each insert and are not
floating or moving.

Figure 3. Slice treatment and dissection of the hippocampus. A. Slice recovery and treatment
set-up. B. A single slice is shown on the Integrid Petri dish before
dissection of the hippocampus. Hippocampus is outlined by a dashed line
in the middle of the slice.

After recovery, the slices are
ready for experimental treatment. Treat slices in oxygenated regular
aCSF either with vehicle or a pharmacological agent of interest.
Treatment times should be determined empirically (seconds to minutes).
Slices from each animal should be distributed evenly between vehicle and
treatment groups. We pool four slices for each sample, which typically
yields 300-400 μg total protein per sample.

After treatment,
using a disposable transfer pipette, immediately transfer slices into
the Integrid Petri dish with ~20 ml of 0.2 N perchloric acid solution
made from 70% perchloric acid in regular aCSF to stabilize IP3 and
incubate at room temp for 2 min. Quickly replace perchloric acid-aCSF
with regular aCSF and place the Integrid Petri dish on ice.

Homogenize the tissue on ice using a sonicator (2 times, 3 sec pulses,
using the 40% amplitude setting), keeping the microcentrifuge tube
steady and the sonicator tip submerged half way into the liquid in order
to avoid bubbling.

Measure protein concentration in each
sample using a Bradford assay. Briefly, prepare standards with known BSA
(1 mg/ml stock) concentrations and unknown samples in double distilled
water. Add Bradford protein assay reagent and incubate at room temp for 5
min. Measure absorbance at 595 nm using a spectrophotometer. Build a
standard curve using absorbance values from the standards and determine
protein concentration for each sample based on the standard curve.

Adjust protein concentration in each sample to the same value (e.g., 2
μg/μl) using ice cold HEPES homogenization buffer with protease
inhibitors.

Measuring IP3 levels

Spray down the bench surface with 70% ethanol and clean it thoroughly.
Make sure to carry out the IP3 assay in a dust free environment.

Pipette 10 μl of the blank,
standards, and each sample (20 μg protein per sample) in triplicate into
wells of the 384-well assay plate with non-binding surface using
barrier tips.

Add 10 μl IP3 tracer to each well, followed by 20
μl IP3 binding protein and mix well by pipetting up and down several
times.

Carefully examine the plate for bubbles. If bubbles form,
pop them with barrier pipette tips. Avoid cross-contamination by using a
new tip for each well. Incubate the plate for 30 min at room temp in the
dark on a rotator with gentle mixing (~60 rpm).

Read the
fluorescence polarization signal of the IP3 tracer with a multimode
microplate reader and a fluorescence polarization filter, using 485 nm
excitation and 530 nm emission wavelengths.

Plot a standard curve
and calculate the IP3 concentration in each sample (nM range in the
hippocampus). In addition to absolute concentration values, IP3 levels
for each vehicle and experimental group can also be reported relative to
baseline (no treatment control).

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